DESCRIPTION (Applicant's abstract): Regional deficits of monoamine transmitters are thought to play a critical role in the pathogenesis of various mental diseases, hence it is necessary to have detailed knowledge of the mechanisms underlying the secretion of these agents. The secretory cycle in neurons and neuroendocrine cells involves the release of neurotransmitters and hormones by exocytosis followed by recovery of vesicular membrane by endocytosis. We recently analyzed a novel form of endocytosis, called rapid endocytosis (RE), which occurs in a variety of secretory cells including neurons and is tightly coupled to secretion. RE is distinct from receptor-mediated endocytosis (RME) as it is a much faster process and does not involve clathrin. The protein dynamin is thought to participate in the vesicle fission reaction that is critical to various forms of endocytosis. Multiple forms of dynamin exist, and in this proposal we seek to establish if different isoforms are involved in distinct types of endocytosis as well as determine which domains of dynamin are essential to the function of the protein. For these studies we propose to use the PC12 cell line, a pheochromocytoma that exhibits properties of both adrenal chromaffin (AC) cells and sympathetic neurons. First, we will demonstrate that PC12 cells provide a useful model system for the molecular analysis of the secretory cycle in neuroendocrine cells. We will characterize the kinetics of exocytosis and endocytosis in these cells and assess whether the properties of these processes resemble those previously seen in AC cells. Second we will determine the dynamin isoforms expressed in these cells and their role in RE and RME. For this purpose we will employ a battery of molecular and immunological procedures, including isoform-specific antibodies. Thirdly, we will dissect which dynamin domains are essential for RE and RME, focussing on the pleckstrin-homology (PH) domain and the proline-rich C-terminal. We will introduce a variety of dynamin constructs into PC12 cells followed by measurement of RE and RME. In these studies we will use state-of-the-art electrophysiological recordings including measurements of membrane capacitance to directly monitor exocytosis and RE and amperometric detection of catecholamine release from single cells. These studies will have significance for secretory biology in general and should further our understanding of the cellular mechanisms of monoaminergic transmission so necessary for the development of novel therapeutic strategies.